Precision small volume fluid processing apparatus and method

ABSTRACT

A high precision, small volume fluid processing system employs open ended capillary tubes to meter, aliquot and mix small volumes of sample fluid and reagents. The system has an automatic mechanism for moving the capillary tubes as well as automated sub-systems for incubating and mixing fluids within the capillary tubes.

This application is a divisional application of U.S. patent applicationSer. No. 08/531,215, filed Sep. 19, 1995, and entitled “Precision SmallVolume Fluid Processing Apparatus and Method,” now U.S. Pat. No5,785,926.

TECHNICAL FIELD

The invention relates to methods and apparatus for precisely handlingsmall volumes of fluids. More specifically, the invention relates tomethods and apparatus for aliquoting and assaying biological fluidsamples.

BACKGROUND OF THE INVENTION

Diagnostic and other biological assays often require systems formetering dispensing and mixing reagents with sample fluids. The samplefluids may include, for example, patient samples, blood samples, orminute quantities of deoxygenated rybo nucleic acid (hereinafter “DNA”)sequences in a buffer fluid. Both manual and automated systems have beenavailable for aliquoting the fluid samples, and assaying the sampleswith one or more reagents. Manual systems have historically included theglass capillary pipette, the micro pipette, precision syringes andweighing equipment. A variety of biological assays have been andcontinue to be conducted with manual equipment of the type described.

Relatively sophisticated microbiological assays including micro-enzymelinked immunosorbent sandwich assays (hereinafter “ELISA”) can besatisfactorily, if tediously performed manually. The demands of modernantibody/antigen matching, histocompatibility typing, paternity testing,etc. on a vast scale has precipitated the development of variousautomated assay equipment to more quickly process large numbers ofpatient samples with various reagents. It is apparent that in order toperform a multiplicity of assays with a single patient sample, theamount of sample must be relatively large, or a small sample must bealiquoted into smaller divisions.

Recent advances in microbiology have provided the biotechnologist withincreasingly sophisticated tools for examining genetic material.Restriction enzyme digestion and polymerase chain reaction (hereafter“RED and PCR respectively”) have provided geneticists with multiple DNAsegments from a single sample for subsequent assaying. All of theseadvances have increased the need for sample handling and processingtechniques which are beyond the ability of the heretofore manualpipetting and other standard laboratory techniques. As a result, theindustry has proceeded with the development of highly automatedequipment which can rapidly and repeatably handle relatively smallquantities of patient samples.

The undertaking of the Human Genome Project exceeds the limits ofcurrent fluid sample handling and processing technology. The HumanGenome Project is an attempt to map the entire human genetic code,nucleotide by nucleotide. The PCR and RED techniques presently availablewill therefore produce an extremely large numbers of nucleotide segmentswhich must be assayed in a variety of different ways. In addition,current methods for producing the nucleotide segments are extremelyexpensive requiring the very wise use of the resultant sample. It iscurrently calculated that without further advancement in the state ofthe art, the cost of producing sufficient samples for laboratoriesaround the world will be prohibitive without the development oftechniques for handling much smaller samples and reagent volumes.

Therefore, a need exists for a high-precision, small volume fluidprocessing system which can aliquot and dispense fluid samples inextremely small volumes, react the samples with small quantities ofreagents, and perform all of the other steps which may be necessary in aconventional assay. The system should also preferably be relativelyhighly automated so that the incidence of human error is reduced.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a highprecision, small volume fluid processing system which can preciselyaliquot small volumes of a sample fluid.

It is further object of the invention to provide a high-precision, smallvolume fluid processing system which can mix small aliquots of samplefluid with various discreet fluid reagents.

It is a further object of the present invention to achieve the aboveobjects in a system which automatically aliquots the fluid sample,introduces appropriate reagents, mixes the sample and reagents, andincubates the same in preparation for gel electro phoresis.

It is yet a further object of the present invention to achieve the aboveobjects in a system which can precisely and repeatably handle fluidvolumes as small as 0.1 μl.

The invention achieves the objects, and other objects and advantageswhich will become apparent from the description which follows byproviding a small volume fluid processing system employing at least onesmall volume capillary tube. A precision linear actuator connected to acomputer controlled motor acts as a pneumatic piston to preciselyinspire and expire one or more fluids into or out of the capillary in apredetermined sequence.

In a preferred embodiment, the fluid processing system can include asample fluid station for containing an initial volume of sample materialin a buffer solution or water, a reagent fluid dispensing device,monitoring equipment for determining the position of a fluid segment inthe capillary tube and a mechanism for precisely positioning the fluidsample handling device in a reference plane with respect to the reagentfluid dispensing device in the sample fluid station. The sample fluidhandling device can intake a precise volume of sample fluid from thesample fluid station, position itself adjacent to the reagent fluiddispensing device which can dispense an appropriate reagent into an openend of the capillary tube. The monitoring device can provide informationto a computer or other management system to either advance or retard theprecision linear actuator so as to move the fluids appropriately in thecapillary tube. If two or more fluids have been received in thecapillary tube, the linear actuator can be advanced and retarded withdifferential velocities so as to mix the fluids in the capillary tube.Very small volumes of fluids, as little as 0.1 μl can be handles with anaccuracy of ±0.01 μl and similar repeatability if the precision linearactuator is driven by a computer controlled motor rotatably connected toa precision lead screw. The system described above may optionallycontain a heating and cooling system for incubating the capillary tubein a controlled manner. The heating system can include a thin layer ofhighly resistive, transparent material on the outside of the tube whichcan be electrically excited so that a heat generating current flowstherethrough. A fan can be used to cool the capillary tube while it isheated or afterwards to maintain a desired temperature or quickly coolthe tube. The monitoring device for determining the position of a fluidsegment in a capillary tube can be of the optical type including a lightemitter/detector pair or array positioned in proximity to the capillarytube.

The sample fluid handling device can form a bubble or droplet of sample,reagent or other fluid on the open end of the tube having a knownvolume. By positioning a second capillary tube in alignment with andadjacent to the first capillary tube the droplet can be transferred fromthe first tube to the second tube by advancing the tubes towards oneanother until the droplet touches the second tube. Capillary actiondraws the droplet into the second tube in a repeatable manner. In thisway, very small quantities of fluid can be transferred from onecapillary to another. In an aliquoting method employing the processingsystem, the sample fluid handling device can inspirate aliquots of fluidsample separated by air gaps therebetween for a more precise dispensingof the aliquots into separate receiving capillary tubes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an isometric, environmental view of a high-precision, smallvolume fluid processing system of the present invention.

FIG. 2 is a side elevational view of the system shown in FIG. 1.

FIG. 3 is an enlarged, partial side elevational view of a capillary tubeof the sample fluid handling device approaching a sample fluid station.

FIG. 4 is an enlarged, partial side elevational view similar to FIG. 3showing the capillary tube inspiring a precise volume of sample fluid.

FIG. 5 is an enlarged, partial elevational view similar to FIG. 4showing the sample fluid handling device being retracted from the samplefluid station.

FIG. 6 is an enlarged, partial elevational view of a reagent fluiddispensing device projecting reagent fluid droplets into the capillarytube.

FIG. 7 is an enlarged, elevational view of the capillary tube undergoinga mixing action.

FIG. 8 is a view similar to FIG. 7 showing the results of the mixingaction.

FIG. 9 is a schematic representation of a piezo electric reagentdispenser.

FIG. 10 is an enlarged, partial sectional view of the circle area ofFIG. 9.

FIG. 11 is a schematic representation of process steps employed by thesystem of the present invention.

FIG. 12 is a schematic representation of an incubation system for thecapillary tubes.

FIG. 13 is a schematic representation of an optical monitoring devicefor determining the position of fluid segments within the capillarytube.

FIG. 14 is an isometric, environmental view of a system for incubating aplurality of the capillary tubes in a controlled manner.

FIG. 15 is an enlarged, sectional view of a capillary tube employing amixing compartment in a first alternate embodiment thereof.

FIG. 16 is a view similar to FIG. 15 of a second alternate embodiment ofa capillary tube employing a mixing chamber.

FIG. 17 is a partial, sectional elevational view of an adapter forfluidly connecting a capillary tube with a motor driven precision leadscrew mechanism.

FIGS. 18A, 18B and 18C show a schematic representation of a series ofthree steps for transferring a fluid droplet from a dispensing capillaryto a receiving capillary in a method of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A high precision, small volume fluid processing system, in accordancewith the principles of the invention, is generally indicated atreference numeral 20 in the Figures. With reference to FIGS. 1 and 2,the system includes a base 20, an X-Y axis positioning mechanism 24, areagent dispensing station 26 and a sample station 28. The base includesa pair of supports 30 which position a base platform 32 at a 45° anglewith respect to a support surface 34. The sample station 28 and reagentdispenser station 26 are positioned at 90° angles with respect to theplatform 32 thus position the same at another 45° angle, with respect tothe support surface 34. As shown in more detail in FIGS. 3-5, theresulting spacial relationships positions the surface of a sample fluid36 at approximately a 135° angle with respect to an open ended,hydrophilic capillary tube 38 for purposes which will be described infurther detail below.

The X-Y positioning mechanism 24 includes a precision, screw actuatedbed 40 for positioning an X axis stage 44 laterally with respect to thesample station 28 and reagent dispensing station 26. An appropriatedprecision screw actuated bed 40 is available from Applied Precision,Inc., Mercer Island, Wash., under the model number AP 1000. The X axisstage has a Y motion frame 46 adapted for transverse motion with respectthereto so as to position the capillary tube 38 axially with respect tothe sample station 28 and reagent dispensing station 26. This isaccomplished by mounting a stepper motor driven, computer controlled,precision linear actuator 48 on the X axis stage 44, while an axiallyreciprocable portion thereof 49 is mechanically connected to the Ymotion frame 46. A computer or other suitable control device isconnected to the actuator 48 by way of a control cable 50 in a manner tobe described more fully below. An appropriate precision linear actuator48 is available from Applied Precision, Inc., Mercer Island, Wash.,under the brand name Nanomover™.

The Y motion frame 46 supports a sample fluid handling device generallyindicated at reference numeral 52 which can precisely exspirate, andaspirate very small volumes of sample fluids, reagent fluids, etc. Thedevice 52 includes a second, precision linear actuator 54, the openended hydrophilic capillary tube 38 previously described, and an adaptermechanism 56 for fluidly interconnecting the capillary 38 with theactuator 54 such that an internal spindle of the actuator acts as a highprecision, pneumatic piston for controlling the position of a fluidsegments in the capillary tube. A second control cable 58 operativelyinterconnects the precision stepper motor of the actuator 54 with acomputer or other appropriate control device in a manner similar to thatdescribed with respect to the first precision linear actuator 48. Bothactuators contain relative or absolute position sensors 60, 62 whichprecisely determine the position of the stepper motor within theactuators, and thus the precise position of a fluid segment within thecapillary tube 38.

Preferably, the sample fluid handling device 52 has mounted thereon anoptical position determining sensor 64 one portion of which has beenremoved for clarity in FIG. 1 and which is illustrated in furtherdetail, schematically in FIG. 13 to provide a fluid segment positionfeedback loop for directly determining the position of a fluid segmentwithin the capillary tube 38. The sensor 64 is operativelyinterconnected with a computer or other appropriate control devicethrough a third control cable 70.

The system described above permits very small, precise volumes of fluidsample and fluid reagents to be precisely metered, mixed, and incubatedfor advantageous application to PCR, enzyme restriction digestion,ELISA, DNA sequencing, and other microbiological assays and processingtechniques. The system can accurately handle fluid samples as small as0.1 μl with an accuracy of ±0.01 μl.

This technique is further illustrated in FIGS. 3-8 in which the samplestation 28 has been adapted to receive a conventional, 96 wellmicrotiter plate 72 having a plurality of microwells 74 containing avolume (typically 5 ml or less) of a fluid sample 36. The samples can besegments of DNA in a buffer solution from a single individual, or thelike. As shown in FIG. 3, the capillary 38 is positioned laterally bythe precision screw actuated bed 40 so as to be in alignment with one ofthe microwells 74 of the microtiter plate 72. The capillary tube is thenadvanced in the direction of the arrow under the urging of the firstprecision linear actuator 48 until it is in contact with a lower levelof the fluid 36 as shown in FIG. 4. Sample fluid is then aspirated intothe capillary tube 38 both by the natural capillary action of the tube,as well as by withdrawal of a spindle portion of the actuatorfunctioning as a pneumatic piston as best seen with reference to FIG.17.

As shown in FIG. 17, the second, precision linear actuator 54 has areciprocatable spindle portion 80 capable of precise axial movement in amanner well known to those of ordinary skill in the art. The actuator 54has an extremely threaded end section 82 which has received thereon acylindrical adapter 84 preferably manufactured from a nonreactivematerial such as stainless steel or heat treated aluminum. The adapterhas a reduced diameter internal cavity 86 having a diameter ofapproximately 0.25 inch and an axial length of approximately 0.25 inch,and thus a volume of approximately 0.0128 cubic inches. The spindleportion 80 can protrude into this cavity so that the spindle and cavityform a precision pneumatic piston and cylinder. A forward, externallythreaded end portion 88 of the adapter has a small diameter bore 90therethrough for communication with an axial bore 92 of a threaded cap94. The cap is preferably manufactured from Delrin, or another suitablynonreactive thermoplastic material. A small diameter washer 96, and “O”ring 98 are seated on an interface of the adaptor and cap so as tosealingly receive and seat the hydrophilic capillary tube 38 and toprovide fluid communication between the capillary tube and bore 90. Aradially directed vent 100 is in fluid communication with the axial bore92 to relieve pressure within the cavity 86, bore 90 and bore 92 whenthe capillary tube is inserted into the cap 94.

With reference again to FIGS. 3, 9, and 8 is now apparent that uponretracting the spindle portion 80 in FIG. 17, a controlled volume ofsample fluid 36 will be drawn into the capillary tube 38 so as to form asample fluid segment 112 as shown in FIG. 5 when the capillary tube iswithdrawn from the microtiter plate 72. As further shown in FIG. 5, theposition of the sample fluid segment 112 within the tube can becontrolled by appropriate operation of the second precision linearactuator 54. In this way, an air gap 114 may or may not be created so asto form a physical barrier between the segment 112 and any additionalsample fluid segments which may be inspired into the capillary tube 38for subsequent aliquoting purposes as shall be described further hereinbelow.

A suitable capillary tube 38 has a length of approximately 55millimeters, a maximum interior volume of 5 μl. The capillary has aninner diameter of 0.0134 inches. Tubes of this type are available fromDrummond Scientific Company, Broomall, Pa., under the trademarkMICROCAPS, part no. 1-00-0050-55. Capillary tubes of this type arenaturally hydrophilic when clean. Thus, upon inserting the capillarytube 38 into the sample fluid 36 as shown in FIG. 4, a small amount offluid sample will be drawn into the capillary without any movement ofthe spindle portion 80 of the second precision linear actuator 54. Inorder to compensate for this “dead volume”, a correction factor must beapplied to the command signal sent to the second actuator through thecable 58. The dead volume V_(d) follows the formula:

V _(i) =m V _(d) +b.

The dead volume V_(d) may be calculated as: V _(d) =X _(piston) k+V_(d-min), where X_(piston) represent the position of spindle portion 80,k represents a conversion factor from linear spindle position todisplaced volume, and V_(d-min) represents the minimum dead volumeinherent in the system. In order to aspirate a desired volume of samplefluid 36, (V_(a)), the commanded aspiration is V _(a-command) =V _(a) -V_(l).

Using five μl capillaries and the Nanomover™ brand linear actuator 52,adapter 56 and cap 94 described above, the quantities have the followingvalues.

Quantity Value m 5.11 × 10⁻³ b 0.057 μl k 31.7 μl/mm V_(d-min) 28 μl

This, for initial position of X_(piston)=0, the dead volume, V_(d) is 28μl and the initial take up due to capillary action is V_(l)=0.2 μl.Therefore, for commanded aspiration of 0.1 μl of sample fluid 36, thistranslated into a commanded aspiration of V_(a-command)=−0.1 μl—anegative aspiration command. That is, in order to compensate for thecapillary action of hydrophilic tube 38, spindle portion 80 must moveforward a distance equivalent to a volume of 0.1 μl. Larger desiredvolumes, V_(a) will of course require positive aspiration commands.

It is possible to avoid the dead volume problem described above by usinga hydrophobic capillary tube. The clean glass capillary can be madehydrophobic by coating the interior thereof with silicone oxide oranother material well known to those of ordinary skill in the art. Asuitable product is available from Sigma Chemical Co., St. Louis, Mo.,under the trademark SIGMACOTE, part no. SL-2.

Once the desired volume of sample has been aspirated into the capillarytube 38 as shown in FIGS. 3-5, the capillary tube 38 can be laterallymoved into alignment with respect to the reagent dispensing station 26to receive one or more appropriate reagents into the capillary tube forsubsequent mixing with the fluid sample segment 112 of FIG. 5. Thepreferred embodiment of the invention employs piezo electric reagentdispensers 120 as shown in greater detail in FIGS. 9 and 10. Anappropriate reagent dispenser of this type is disclosed by Hayes, et al.in U.S. Pat. No. 4,877,745 the disclosure of which is incorporatedherein by reference. It is sufficient for the purposes of thisdisclosure to explain that the piezo electric reagent dispenser has areagent well 122 closed by a removable cap 124. The well 122 contains anappropriate reagent 123 and is fluidly connected to a dispensing tube126 having a dispensing orifice 128 at a free end thereof. Thedispensing tube is concentrically surrounded by a piezo electric element130 which when stimulated by an appropriate voltage generates aparistalic acoustic wave within the tube ejecting one or more droplets132 of a reagent of known volume in a highly precise manner.

As shown in FIG. 6, the sample fluid segment 112 should be withdrawn adistance which corresponds to the volume of the droplets 132 andsimultaneously with the expulsion thereof from the dispensing tube 126so that the droplets do not encounter back pressure within the capillarytube. The result will be a second fluid segment 134 consistingexclusively of reagent fluid adjacent to the sample fluid segment 112.If an air gap 114 as shown in FIG. 5 is desired the series of segmentswithin the capillary tube 38 shown in FIG. 7 will be as follows: reagentfluid segment 134, air gap segment 114, and the sample fluid segment112. In either event, by oscillating the segments within the capillarytube 38 in opposite directions with differential velocities V₁, V₂ thefluids will mix within the capillary tube. To ensure adequate mixing,one of the velocities should be at least three times the other velocity,and the mixing should occur over 100 cycles at a frequency of threecycles per second. In a five μl capillary when 3 μl of fluids have beenreceived, axial oscillation of the fluids, of ±1.5 μl can occur withoutinadvertently aspirating the fluids from the free end of the capillarytube 38.

In order to accurately position the fluid segments with the capillarytube 38, an optical position measuring device 64 as shown in FIG. 1 ispreferably provided although all of the sequencing steps described abovecan be and have performed in an open loop mode. The device 64 is furtherdescribed schematically in FIG. 13 wherein a printed circuit board 140supports a charge couple device 142 thereon for sensing the position ofthree exemplary fluid segments 144, 145, and 146 in the capillary tube38. An array of light emitting diodes 148 illuminates the fluid segmentswithin the capillary tube 38. Poto diodes detects the presence of lighttransmitted through air gaps 151, 152, and 153 and outputs thisinformation to a charge coupled device which then transfers thisinformation serially to a digital computer or other appropriate controldevice. As shown in the schematic representation at reference numeral154, a graph of signal amplitude on the vertical axis versus segmentposition with respect to the free end 136 of the capillary tube 38indicates not only the presence or absence of the fluid segments buttheir respective leading and trailing edges as well. Using standardinterpolation techniques, resolutions of up to 50 nanometers of absoluteposition have been achieved by using a charge couple device array 42manufactured by Sony under the model no. IFX503A. A development kit isincluded with this part which will enable those of ordinary skill in theart to execute the design shown in FIG. 13 without undueexperimentation.

With reference to FIG. 11, it is seen that an appropriately programmedpersonal computer 156 employing conventional analog to digitalcontroller modules (not shown) can be operatively interconnected withthe precision screw actuated bed 40, first precision linear actuator 48,second precision linear actuator 54, piezo electric reagent dispensers120 and optical position determining sensor 64 so as to comprise a fullyautomated precision small volume fluid processing apparatus. Inaddition, a capillary dispensing station 160 having a V shaped capillarytrough 162 can be used to position individual blank capillary tubes in areceptacle 164. As will be apparent from examination of FIG. 17, it ispossible for the second linear actuator 48 to advance the sample fluidhandling device linearly towards the receptacle 164 (which is shown inelevational view for clarity) so as to insert a blank capillary tube 38into the cap portion 94. The computer can then command the precisionscrew actuated bed 40 to position the second actuator 54 in alignmentwith the sample station 28 and receive a predetermined volume of sampleas previously described. The optical position determining sensor 64 canthen send position information to the computer 156 to confirm that thecorrect volume of sample fluid has been aspirated and positionedappropriately within the capillary tube 38. The computer can thencommand the bed 40, and actuator 48 to move the capillary tube 38 to bemoved into appropriate positioning 38″ with respect to the reagentdispensing station 26 to receive one or more precisely predeterminedvolumes of reagent. Once the reagent(s) and fluid sample have beenappropriately mixed within the capillary tube 38, the computer cancommand the capillary tube 38 to be moved to a new position 38′″ forfurther processing.

As shown in FIG. 12, further processing can include incubating thecapillary tube 38 in an appropriate temperature controlled environment.This result can be achieved by providing a thin, substantiallytransparent, highly resistive coating 170 on the exterior of thecapillary and connecting electrodes 172, 174 to an appropriate voltagesource 178 which is in operative communication with the computer 156.Appropriate coating is indium tin oxide applied by the conventionalvapor deposition techniques. It is known that the resistivity of a thinfilm of indium tin oxide changes with temperature, thus a continuousresistive measurement to be made by an electronically controlled Ohmmeter 178 also operatively connected with the computer 156 can be usedas a measurement of the tube temperature. A cooling fan 180 ispreferably positioned advantageously with respect to the capillary tube38 provide a constant air flow over the tube and a rapid cool down cyclewhen the voltage is no longer applied across the electrodes 172, 174. Aconventional temperature sensor such as Thermistor type temperaturesensitive resister 182 can also be preferably operatively interconnectedwith the computer to measure the temperature of the air flow exiting thefan 180.

FIG. 14 illustrates a preferred embodiment for implementing theincubation technique shown schematically in FIG. 12. Before theincubation step shown in FIG. 12 can proceed, both the free end 136 andan open mounting end 137 of the tube 38 should be heat sealed in theconventional manner. Once this has been done, a plurality of the sealed,capillary tubes 36 can be incubated by placing them in a holder,generally indicated at reference numeral 190 in FIG. 14. The holderincludes opposed end caps 192, 193 having the electrodes 172, 174therein and in communion with appropriately sized apertures 194 forreceiving the capillary tubes 36. The exterior ends of the capillarytubes are preferably plated with chrome or another conductive materialto make good electrical contact with a peripheral copper strip 196, 197connected to the electrodes 172, 174. A space block 200 provides amechanical support for the end caps to prevent the tubes from beingcrushed. In this preferred embodiment, the cooling fan 180 andThermistor™ are contained in a plenum or duct 210 which directs thecooling air over and through the capillary tubes 36 for the purposespreviously described.

As described above, the capillary tube 38 is preferably a hydrophilictube. Moreover, as previously stated, the tube may be made hydrophobicby the introduction of appropriate materials. FIG. 15 shows an alternatepreferred embodiment of a hydrophobic capillary tube 220 having anenlarged diameter mixing chamber 222. In this embodiment, the enlargedchamber in combination with an interior hydrophobic surface 224increases turbulence in the tube during mixing and thus improves themixing process. One technique for manufacturing such a tube is shown inFIG. 16 in another alternate embodiment in which two conventionalsections of hydrophilic capillary tube 38 are joined in a spaced apartrelationship by a larger diameter section 230 of a hydrophobic capillarytube section.

In the case of either embodiment shown in FIG. 15 or 16, these capillarytubes can be used in the same manner as shown with respect to thepreferred, hydrophilic tube. Finally, it should be noted that acapillary tube which is neither hydrophilic or hydrophobic can furtherbe used.

FIG. 18 illustrates an aliquoting method using the system shown in FIG.10 wherein the reagent dispensing station 26 is provided with one ormore hydrophilic receiving capillary 300 in place of the reagentdispensers 120. In this method as shown in FIG. 18(a) one or moresegments 112 of sample fluid have been inspired into the capillary tube38 and are separated by an air gap 114. By advancing the spindle portion80 a desired amount a droplet 310 of known volume can be formed at thefree end 136 of the capillary tube 38. As an example of the preferredembodiment given above, each step taken under computer control by thestepper motor of the actuator 54 corresponds to an increase in the sizeof the droplet 310 of 1.583 nanoliters. When an appropriately sizeddroplet is formed, the capillary tube 38 is advanced towards thereceiving tube 300 by the first linear actuator 48 as shown in FIGS. 1and 2. As shown in FIG. 18(b) as soon as the droplet contacts the freeend 320 of the receiving capillary 300 the droplet 310 of known volumewill be drawn into the receiving capillary 300 by capillary action. Thesteps shown in FIG. 18(a) and 18(b) can be repeated until a sample fluidsegment 330 having a volume equal to or less than the fluid segment 112has been transferred. Droplets as small as 50 nanoliter increments havebeen transferred using this technique with the capillaries described.Once all of the sample fluid segment 112 has been transferred, the airgap 114 prevents the next sample fluid segment is capillary 38 frombeing inadvertently transferred. The capillary 38 may then be laterallymoved to a second receiving capillary (not shown) and the process may berepeated. In this manner, the capillary tube 38 can be used to aliquotDNA samples into very small volumes, and distribute those volumes intoseparate receiving capillary tubes 300 which may then be individuallyprocessed with the appropriate as described herein above.

EXAMPLE I

The apparatus described above has been used to perform a restrictionenzyme digest as described below.

In restriction enzyme digest (RED), a DNA sample is mixed with a“restriction enzyme” in a buffered solution and incubated. Eachrestriction enzyme will cleave that DNA at each site containing thesequence of base pairs specific to that enzyme. Restriction enzymes aresold commercially. An enzyme and DNA segment were chosen known toproduce multiple cuts.

The DNA fragment sizes are determined by gel electrophoresis after theincubation. In this procedure, the (digested) DNA is placed on a gel(typically agarose) which is immersed in an electric field. The DNA willmigrate through the gel at a rate determined by it's size: big piecesare slower.

First, an enzyme solution was prepared containing “Hind III” restrictionenzyme, a buffer sold with the enzyme which is specific to that enzyme,sterile water, and an extra protein known as “BSA”, according to thefollowing recipe:

15 parts sterile, double distilled water

2 parts buffer

1 part BSA (2.5 mg/μl)

1 part enzyme (Hind III)

The BSA is necessary to keep the enzyme in the solution. Enzymes, likeall proteins, tend to adhere to surfaces such as the inside of capillarytubes, test tubes, the pipette tips, and the reagent dispensers 120. TheBSA is used as a sacrificial protein to occupy the adhesion sites andhelp keep the enzyme in solution. Gelatin and BSA are both commonly usedfor this purpose in micro-biology/genetics procedures.

The DNA chosen for this experiment was commercially prepared “Lambda”DNA, GibcoBRL part #25250-010, concentration 0.52 ug/μl. The enzymechosen was “Hind III”, GibcoBRL part #15207-012, concentration 10 U/μl,which comes with it's own specific buffer (GibcoBRL “React 2” buffer).

Starting with a clean, sterile container, 1 part DNA was added with 19parts of the enzyme solution listed above. The resulting mixture wasagitated, then incubated at 37 C for two hours. The volumes were reducedusing the method of the invention by a factor of 10, to 0.1 μl of DNAand 1.9 μl of enzyme.

A large batch of the enzyme mix, 200 or 300 μl worth was mixed up withthe excess and used to prime the reagent dispenser 120. The capillarieswere then flame sealed with a butane torch prior to incubation.

Next the samples are removed from their capillary and placed on a 2%,agarose gel of the “high melt” variety—commonly known as “2% HMPagarose”. The gel is exposed to an electric field, around 100-120 volts,for 15-20 minutes.

After electrophoreses, the gel is soaked in an Ethidium Bromide solutionto stain the DNA, and examined under a UV light. DNA will show up asflorescent bands.

All but the two smallest DNA bands could be identified. GibcoBRL note intheir documentation these two bands are hard to distinguish because theyare so much smaller than the largest fragment sizes, and this samecaveat applies to their reference.

The bands from the samples matched the bands in the GibcoBRL reference,with the two smallest bands visible only faintly, even in the reference.The second smallest band was made more visible in the reference whichwas intentionally overloaded a gel lane with excess DNA.

Other embodiments and variations of the invention are contemplated.Those or ordinary skill in the art will indeed conceive of otherembodiments and variations of the invention upon review of thisdisclosure which are not herein described but are within the spirit ofthe invention. For example, it is contemplated that the receivingcapillary 300 can be hydrophobic and can have an open mounting end (notshown) fluidly connected to a third precision linear actuator, similarto linear actuators 48, 54, by way of an additional adaptor 84 and cap94. The third linear actuator can be coordinated with the first actuatorwhich drives the dispensing capillary 38 in FIG. 18(a). In this way, thedroplet 310 can be aspirated into the receiving capillary under positivecontrol rather than by capillary action. Therefore, the invention is notto be limited by the above disclosure, but is to be determined in scopeby the claims which follow.

What is claimed is:
 1. A method of mixing discrete fluid segments in aconstant-diameter, straight capillary tube that has first and secondopen ends, comprising: introducing a first fluid volume into the firstend of the capillary tube, so as to form a first fluid segment withinthe tube; introducing a second fluid volume into the first end of thecapillary tube, while simultaneously drawing a partial vacuum at thesecond end of the capillary tube, so as to move the first fluid segmentfurther into the capillary tube and form a second fluid segment withinthe capillary tube, separated from the first fluid segment by an airgap; and oscillating the fluid segments within the capillary tube inopposite directions and at different velocities to cause the fluidsegments to mix within the capillary tube.
 2. The method of claim 1,wherein the first fluid segment is a sample fluid segment and the secondfluid segment is a reagent fluid segment.
 3. The method of claim 1,comprising oscillating the fluid segments within the capillary tube by avelocity V₁ in a first direction, and a velocity V₂ in the oppositedirection, wherein velocity V₁ is at least approximately three timesvelocity V₂.
 4. The method of claim 1, comprising connecting thecapillary tube to a precision linear actuator and operating theprecision linear actuator to oscillate the fluid segments within thecapillary tube.
 5. The method of claim 4, comprising using a computer tooperate the precision linear actuator.
 6. The method of claim 1,comprising using a hydrophilic capillary tube.
 7. The method of claim 6,wherein the first fluid segment is a sample fluid segment and the secondfluid segment is a reagent fluid segment.
 8. The method of claim 6,comprising oscillating the fluid segments within the capillary tube by avelocity V₁ in a first direction, and a velocity V₂ in the oppositedirection, wherein velocity V₁ is approximately three times velocity V₂.9. The method of claim 6, comprising connecting the capillary tube to aprecision linear actuator and operating the precision linear actuator tooscillate the fluid segments within the capillary tube.
 10. The methodof claim 9, comprising using a computer to operate the precision linearactuator.